EP2534245A2 - Optmierte zellulaseenzyme - Google Patents

Optmierte zellulaseenzyme

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Publication number
EP2534245A2
EP2534245A2 EP11706777A EP11706777A EP2534245A2 EP 2534245 A2 EP2534245 A2 EP 2534245A2 EP 11706777 A EP11706777 A EP 11706777A EP 11706777 A EP11706777 A EP 11706777A EP 2534245 A2 EP2534245 A2 EP 2534245A2
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EP
European Patent Office
Prior art keywords
seq
polypeptide
amino acid
sequence
positions
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Granted
Application number
EP11706777A
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English (en)
French (fr)
Other versions
EP2534245B1 (de
Inventor
Ulrich Kettling
Christoph Reisinger
Thomas Brück
Andre Koltermann
Jochen Gerlach
Isabel Unterstrasser
Lutz Röcher
Markus Rarbach
Jörg CLAREN
Andreas Kohl
Jan Carsten Pieck
Dominik Schlosser
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
Sued Chemie IP GmbH and Co KG
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Sued Chemie IP GmbH and Co KG
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Priority to SI201131575T priority Critical patent/SI2534245T1/sl
Priority to PL11706777T priority patent/PL2534245T3/pl
Priority to RS20181132A priority patent/RS57695B1/sr
Priority to EP11706777.7A priority patent/EP2534245B1/de
Publication of EP2534245A2 publication Critical patent/EP2534245A2/de
Application granted granted Critical
Publication of EP2534245B1 publication Critical patent/EP2534245B1/de
Priority to HRP20181587TT priority patent/HRP20181587T1/hr
Active legal-status Critical Current
Anticipated expiration legal-status Critical

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Classifications

    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/14—Hydrolases (3)
    • C12N9/24—Hydrolases (3) acting on glycosyl compounds (3.2)
    • C12N9/2402—Hydrolases (3) acting on glycosyl compounds (3.2) hydrolysing O- and S- glycosyl compounds (3.2.1)
    • C12N9/2405—Glucanases
    • C12N9/2434—Glucanases acting on beta-1,4-glucosidic bonds
    • C12N9/2437—Cellulases (3.2.1.4; 3.2.1.74; 3.2.1.91; 3.2.1.150)
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/14—Hydrolases (3)
    • C12N9/24—Hydrolases (3) acting on glycosyl compounds (3.2)
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Y—ENZYMES
    • C12Y302/00—Hydrolases acting on glycosyl compounds, i.e. glycosylases (3.2)
    • C12Y302/01—Glycosidases, i.e. enzymes hydrolysing O- and S-glycosyl compounds (3.2.1)
    • C12Y302/01091—Cellulose 1,4-beta-cellobiosidase (3.2.1.91)
    • Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
    • Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
    • Y02E—REDUCTION OF GREENHOUSE GAS [GHG] EMISSIONS, RELATED TO ENERGY GENERATION, TRANSMISSION OR DISTRIBUTION
    • Y02E50/00—Technologies for the production of fuel of non-fossil origin
    • Y02E50/10—Biofuels, e.g. bio-diesel

Definitions

  • the invention discloses cef!ulase enzymes with optimized properties for processing of cellulose- and lignoceltulose-containing substrates.
  • cellobiohydrolase enzymes with preferred characteristics are disclosed.
  • the present invention provides fusion, insertion, deletion and/or substitution variants of such enzymes. Enzyme variants have enhanced thermostability, proteolytic stability, specific activity and/or stability at extreme pH.
  • Nucleic acid molecules encoding said enzymes, a composition comprising said enzymes, a method for preparation, and the use for cellulose processing and/or for the production of biofuels are disclosed.
  • Cellulose material in pure form or in combination with hemicellulose and/or iignin is a valuable and readily available raw material for the production of chemicais and fuels.
  • a key step in processing cellulose and iignoceilulose is the hydrolysis of the beta-1 ,4-linked glucose polymer cellulose and the subsequent release of glucose monomers and short glucose oligomers such as cellobiose, cellotriose, etc. Enzymes that catalyze this reaction are found in various organisms, especially filamentous fungi and bacteria, that are capable of degrading and hydrolysing cellulose .
  • Treatment to make ceilulosic substrates more susceptible to enzymatic degradation comprises milling, chemical processing and/or hydrothermal processing. Examples are wet oxidation and/or steam explosion. Such treatments increase the accessibility of cellulose fibers and separate them from hemice!luiose and iignin.
  • CBH cellobiohydrolase
  • CBHI cellobiohydrolase I
  • Hydrolyzed celSuiosic materials contain several valuable carbohydrate molecules which can be isolated from the mixtures.
  • Sugar containing hydrolysates of cellulosic materials can be used for microbial production of a variety of fine chemicals or biopolymers, such as organic acids, ethanol or higher alcohols (also diols or polyols) or polyhydroxyalkanoates (PHAs).
  • PHAs polyhydroxyalkanoates
  • Kurabi et al. describes preparations of cellulases from Trichoderma reesei and other fungi, such as Penictllium sp. The performance has been analysed on steam-exploded and ethanol organosolv-pretreated Douglas-fir. Better performance of enzyme mixtures appears to be a result of improved properties of singie component enzymes as well as the effect of each compound in the mixture, especially the presence of beta-glucostdase.
  • Kurabi A, Berlin A, Gilkes N, Ki!bum D, Bura R, Robinson J, Markov A, Skomarovsky A, Gusakov A, Okunev O, Sinitsyn A, Gregg D, Xie D, Saddler J. (2005) Enzymatic hydrolysis of steam- exploded and ethanol organosolv-pretreated Dougias-Fir by novel and commercial fungal cellulases. Appl Biochem Biotechnol.121 -124: 219-30).
  • glucohydroiase class 7 (cel7) are known to the art from several fungal sources.
  • the Talaromyces emersonii Cel7 cellobiohydrolase is known and expression was reported in Escherichia coli (Grassick A, Murray PG, Thompson R, Collins CM, Byrnes L, Birrane G, Higgins TM, Tuohy MG.
  • Escherichia coli Gramsick A, Murray PG, Thompson R, Collins CM, Byrnes L, Birrane G, Higgins TM, Tuohy MG.
  • CBH IB Three-dimensional structure of a thermostable native cellobiohydrolase, CBH IB, and molecular characterization of the cel7 gene from the filamentous fungus, Talaromyces emersonii.
  • WO03/000941 discloses a number of CBHs and their corresponding gene sequences. Physiological properties and applications however were not disclosed. The fusion of cellulose binding domains to catalytic subunits of cellobiohydrolases is reported to improve the hydrolytic properties of proteins without a native domain.
  • US 2009042268 discloses fusions of Thermoascus aurantiacus Cel7A with cellulose binding domains from DCiobiohydrolase I from Chaetomium thermophilum and Hypocrea jecorina.
  • Hong et ai. (2003) describe the production of Thermoascus aurantiacus CBHI in yeast and its characterization. (Hong J, Tamaki H, Yamamoto K, Kumagai H Cloning of a gene encoding thermostable DCiobiohydrolase from Thermoascus aurantiacus and its expression in yeast. Appi Microbiol Biotechnol. 2003 Nov;63(1 ):42-50).
  • Tuohy et aL (2002) report the expression and characterization of Talaromyces emersonii CBH. (Tuohy MG, Walsh DJ, Murray PG, Claeyssens M, Cuffe MM, Savage AV, Cough!an MP.:Kinetic parameters and mode of action of the cellobiohydrolases produced by Talaromyces emersonii. Biochim Biophys Acta. 2002 Apr 29;1596(2):366-80).
  • Nevoigt et a!. (2008) reports on the expression of cellulolytic enzymes in yeasts. (Nevoigt E. Progress in metabolic engineering of Saccharomyces cerevisiae. Microbiol Mol Biol Rev. 2008 Sep;72(3):379-412).
  • Cellobiohydrolase i (Cel7) was not used in this setup. (Fujita Y, ito J, Ueda M, Fukuda H, Kondo A. Synergistic saccharification, and direct fermentation to ethanol, of amorphous cellulose by use of an engineered yeast strain codisplaying three types of cellulolytic enzyme. Appl Environ Microbiol. 2004 Feb;70(2):1207-12).
  • Boer H et a! describes the expression of GH7 classified enzymes in different yeast hosts but expressed protein levels were iow.
  • Kanokratana et ai (2008), Li et a! (2009) as well as CN01757710 describe the efficient expression of Cel7 CBH I enzymes, however these proteins are lacking celllulose binding domains required for efficient substrate processing.
  • thermostable cellobiohydrolase from the thermophilic fungus Chaetomium thermophilum and its expression in Pichia pastoris. J Appl Microbiol. 2009 Jun;106(6):1867-75).
  • Voutiiainen (2008) and Viikari (2007) disclose Cel7 enzymes comprising thermostable celiobiohydrolases, however with only low to moderate expression levels from Trichoderma reesei.
  • Voutiiainen SP Puranen T, Siika-Aho M, Lappalainen A, Alapuranen M, Kallio J, Hooman S, Viikari L, Vehmaanpera J, Koivu!a A. Cioning, expression, and characterization of novel thermostable family 7 celiobiohydroiases. Biotechnol Bioeng. 2008 Oct 15;101 (3):515-28.
  • Grassick et al. (2004) disclose unfolded expression of Cellobiohydrolase I from Taiaromyces emersonii in Escherichia coli but not in yeast.
  • Grassick A Murray PG, Thompson R, Collins CM, Byrnes L, Birrane G, Higgins TM, Tuohy MG.
  • CBH IB Three-dimensional structure of a thermostable native cellobiohydrolase, CBH IB, and molecular characterization of the cel7 gene from the filamentous fungus, Taiaromyces emersonii. Eur J Biochem. 2004 Nov;271 (22):4495-506).
  • the present invention provides a polypeptide having cellobiohydrolase activity.
  • the invention provides a thermostable polypeptide having cellobiohydrolase activity. That is, in this embodiment, the polypeptide maintains 50 % of its maximum substrate conversion capacity when the conversion is done for 60 minutes at 60 °C or higher, preferably 62 °C or higher, and in a particular embodiment 64 °C or higher, such as 66 °C or higher.
  • This polypeptide comprises an amino acid sequence with at ieast 54 %, preferably at least 56 %, more preferably at Ieast 58 %, particularly preferably at ieast 60 %, such as at Ieast 62 %, particularly at Ieast 64 %, such as at least ' 66 %, and most preferably preferably at Ieast 68 % sequence identity to SEQ ID N05.
  • the invention also provides a polypeptide which comprises an amino acid sequence with at Ieast 85 % sequence identity to SEQ ID NO: 2.
  • the present invention discloses a nucleic acid encoding the polypeptide of the present invention, preferably having at Ieast 95 % identity to SEQ ID NO: 1 , a vector comprising this nucleic acid and a host transformed with said vector.
  • the present invention further provides a method of producing a celiobiohydrolase protein encoded by a vector of the present invention, a method for identifying polypeptides having celiobiohydrolase activity, and a method of preparing such polypeptides having celiobiohydrolase activity. It also provides a method of identifying such polypeptides which maintain 50 % or more of maimum substrate conversion capacity at elevated temperatures, such as at 60 °C or more.
  • the present invention also provides a polypeptide having celiobiohydrolase activity, wherein the polypeptide comprises an amino acid sequence having at least 85 % sequence identity to SEQ ID NO: 2 wherein one or more specific amino acid residues of the sequence defined by SEQ !D NO: 2 are modified by substitution or deletion, as well as insertion mutants.
  • mutants include Q1 , G4, A6, T15, 028, W40, D64, E65, A72, S86, K92, V130, V152, Y155, K159, D181 , E183, N194, D202, P224, T243, Y244, I277, K304, N310, S31 1 , N318, D320, T335, T344, D346, Q349, A358, Y374, A375, T392, T393, D410, Y422, P442, N445, R446, T456, S460, P462, G463, H468 and/or V482 of amino acids 1 to 500 of SEQ ID NO: 2, but the invention is by no means limited to these. Further specific positions are given below.
  • the present invention provides a polypeptide having celiobiohydrolase activity, which is obtainable by the method of preparing a polypeptide having celiobiohydrolase activity according to the present invention, and a polypeptide having celiobiohydrolase activity, wherein the poiypeptide comprises an amino acid sequence having at least 80 % sequence identity to SEQ ID NO: 5, wherein one or more of the following amino acid residues of the sequence defined by SEQ ID NO: 5 are modified by substitution or deletion, as well as insertion mutants.
  • mutants include Q1 , G4, A6, T15, Q28, W40, D64, E65, A72, S86, K92, V130, V152, Y155, K159, D181 , E183, N194, D202, P224, T243, Y244, I277, K304, N310, S31 1 , N318, D320, T335, T344, D346, Q349, A358, Y374, A375, T392, T393, D410 and/or Y422 of amino acids 1 to 440 of SEQ ID NO: 5, but the invention is by no means limited to these. Further specific positions are given videow.
  • the present invention furthermore provides a poiypeptfde having celiobiohydrolase activity comprising an amino acid sequence having at least 85 % sequence identity to SEQ ID NO: 12 wherein one or more of the following amino acid residues of the sequence defined by SEQ ID NO: 12 are modified by substitution or deletion as well as insertion mutants.
  • mutants include Q1 , T15, Q28, W40, C72, V133, V155, Y158, T162, Y247, N307, G308, E317, S341 , D345, Y370, T389, Q406, N441 , R442, T452, S456, P458, G459, H464 and/or V478, but the invention is by no means limited to these. Further specific positions are given below.
  • the present invention further provides the use of a polypeptide or the composition of the present invention for the enzymatic degradation of lignocellulosic biomass, and/or for textiles processing and/or as ingredient in detergents and/or as ingredient in food or feed compositions.
  • Figure 1 Restriction Maps of pV1 for constitutive expression of Proteins in Pichia pastoris: pUC19 - o : Origin of replication in E. coli; KanR: Kanamycine/G418 Resistance with TEF1 and EMZ Promoter sequences for selection in Pichia pastoris and E. coli, respectively; 5'GAP: glyceraldehyde-3- phosphate dehydrogenase Promoter region; 3'-GAP: terminator region; SP MFalpha: Saccharomyces cerevisiae mating factor alpha signal sequence; MCS: multiple cloning site.
  • KanR Kanamycine/G418 Resistance with TEF1 and EMZ Promoter sequences for selection in Pichia pastoris and E. coli, respectively
  • 5'GAP glyceraldehyde-3- phosphate dehydrogenase Promoter region
  • 3'-GAP terminator region
  • SP MFalpha Saccharomyces cerevisiae
  • FIG. 2 Commassie stained SDS-PAGE of 10-fold concentrated supernatants of shake- flask cultures of Pichia pastoris CBS 7435 containing expression plasmids with coding sequences for the mature CBHI proteins of Trichoderma viride (CBH-f; lane 1 ), Humicola grisea (CBH-d; lane 2), Talaromyces emersonii (CBH-b; lane 3), Thermoascus aurantiacus (CBH-e; lane 4), as well as the Talaromyces emersonii CBHI-CBD fusion (CBH-a; lane 6) and the Humicola grisea-CBD fusion (CBH-g; 7) in N-terminal fusion to the signal peptide of the Saccharomyces cerevisiae mating factor alpha under control of the Pichia pastoris glyceraldehyde-3-phosphate dehydrogenase promoter
  • FIG. 3 Map of the pV3 expression plasmid for protein expression in Pichia pastoris.
  • Replicons pUC19 - oh: Origin of replication in E. coli;
  • ZeoR Zeocine resistance gene with TEF1 and EM7 Promoter promoter sequences for expression in Pichia pastoris and E. coli, respectively;
  • AOX I promoter Promoter region of the Pichia pastoris alcohol oxidase I gene;
  • AOX 1 transcriptional terminator terminator region; SP MFalpha: Saccharomyces cerevisiae mating factor alpha signal sequence; MCS: multiple cloning site.
  • FIG. 4 SDS-PAGE analysis of culture supernatant samples taken from the fermentation of a Pichia pastoris strain with a genomic integration of an AOXI-expression cassette, expressing the Talaromyces emersonii CBHI / Trichoderma reesei -CBD fusion peptide (CBH-a) in a 7I bioreactor during methanol induction. Samples P1 - P7 were are taken at the
  • Figure 5 Map of pV4 expression plasmid for the expression of the Talaromyces emersonii CBHI / Trichoderma reesei -CBD fusion peptide (CBH-ah) in Trichoderma reesei.
  • Replicon pUC19 for replication in E. coli.
  • cbhl 5' 5' promoter region of the Trichoderma CBHI gene; cbhl signal peptide: Coding sequence for the Trichoderma reesei CBHI leader peptide; CBH-a: Talaromyces emersonii CBHI / Trichoderma reesei -CBD fusion peptide: coding region for SeqID NO. 18; cbhl Terminator: 3' termination region of the Trichoderma reesei CBHI locus; hygromycine resistance: coding region of the hygromycine
  • Trichoderma reesei phosphoglycerate kinase promoter cbhl 3': homology sequence to the termination region of the Trichoderma reesei CBHI locus for double crossover events.
  • FIG. 6 SDS-Page of Trichoderma reesei culture supernatants.
  • Lane 1 shows the expression pattern of a replacement strain carrying a Talaromyces emersonii CBHI / Trichoderma reesei -CBD fusion (CBH-ah) inplace of the native CBHI gene.
  • lane 2 shows the pattern for the unmodified strain under same conditions.
  • M molecular size marker
  • the fluorescence values were normalized according to figure 8 over the temperature range from 55°C to 75°C
  • Figure 9 Glucose yields of hydrolysis of pretreated straw with wt and mutated Talaromyces emersonii CBHI / Trichoderma reesei -CBD (CBH-ah) fusion protein after hydrolysis for 48 hours in the presence of a ⁇ -glycosidase.
  • the variants are characterized by the following mutations with respect to SeqID NO. 18 and were expressed from Pichia pastoris in shake flask cultures and isolated from the supernatant by affinity chomatography using Ni-NTA.
  • Figure 10 Alignment of SeqID. NO 2 with the Trichoderma reesei CBHI.
  • the alignment matrix blosum62mt2 with gap opening penalty of 10 and gap extension penalty of 0.1 was used to create the alignment.
  • the present invention provides polypeptides having cellobiohydrolase activity.
  • the invention provides a thermostable polypeptide having cellobiohydrolase activity.
  • the invention discloses protein variants that show a high activity at high temperature over an extended period of time.
  • the polypeptide of the present invention maintains 50 % of its maximum substrate conversion capacity when the conversion is done for 60 minutes at a temperature of 60 °C or higher.
  • the respective temperature is also referred to as the IT50 value.
  • the IT50 value is preferably 60 °C or higher, but more preferably 62 °C or higher.
  • the polypeptide maintains 50 % of its maximum substrate conversion capacity when the conversion is done for 60 minutes at 60 °C or higher, preferably preferably 62 °C or higher, and in a particular embodiment 64 °C or higher, such as 66 °C or higher. Furthermore, the polypeptide maintains 50 % of its maximum substrate conversion capacity when the conversion is done for 60 minutes at 60 °C or higher, preferably preferably 62 °C or higher, and in a particular embodiment 64 °C or higher, such as 66 °C or higher. Furthermore, the
  • SU BSTITUTE SH EET (RU LE 26) polypeptides of the present invention have preferably an IT50 value in the range of 62 to 80 °C, more preferably 65 to 75 °C.
  • Substrate Conversion Capacity of an enzyme is herein defined as the degree of substrate conversion catalyzed by an amount of enzyme within a certain time period under defined conditions ⁇ Substrate concentration, pH value and buffer concentration, temperature), as can be determined by end-point assaying of the enzymatic reaction under said conditions.
  • Maximum Substrate Conversion Capacity of an enzyme is herein defined as the maximum in Substrate Conversion Capacity found for the enzyme within a number of measurements performed as described before, where only one parameter, e.g. the temperature, was varied within a defined range. According to the present invention, the assay described in Example 8 is used to determine these parameters.
  • This polypeptide comprises an amino acid sequence with at least 54 %, preferably at least 56 %, more preferably at least 58 %, particularly preferably at least 60 %, such as at least 62 %, particularly at least 64 %, such as at least 66 %, and most preferably preferably at least 68 % sequence identity to SEQ ID NO: 5.
  • identity over a sequence length of y residues means that y is a - preferably continuous - portion of the parenteral sequence (in this particular case SEQ ID NO: 5, but the same is true throughout this application, also with respect to other, specifically indicated parenteral sequences which which the sequences of this invention may be compared) which is used as a basis for the comparison of sequence identitiy.
  • sequence identity preferably 200 or more, more preferably 300 or more, even more preferably 400 or more, and most preferably 437 positions of the parental sequence given in SEQ iD NO: 5 are taken into consideration.
  • sequence identity preferably 200 or more, more preferably 300 or more, even more preferably 400 or more, and most preferably 437 positions of the parental sequence given in SEQ iD NO: 5 are taken into consideration.
  • the details of how the percentages of sequence identities are calculated are given below. It should also be noted, that, unless explicitly otherwise specified in this specification, the entire sequence of the parental sequence (such as, in this particular case, SEQ ID NO:5) (i.e. from the first to the last amino acid residue) shall be used as a parent sequence.
  • the polypeptide capable of maintaining 50 % of its maximum substrate conversion capacity when the conversion is done for 60 minutes at 60 °C or higher, preferably preferably 62 °C or higher is a polypeptide which differs from SEQ ID NO: 5 by at least one mutation, wherebey the mutation may be an insertion, deletion or substitution of one or more amino acid residues. Also preferred are at least two such mutations, such as at least 4, at least 5, at ieat 6, at least 7, at least 10 such mutations with respect to the polypeptide given in SEQ ID NO: 5.
  • Cellobiohydrolase or “CBH' 1 refers to enzymes that cleave cellulose from the end of the glucose chain and produce cellobiose as the main product.
  • Alternative names are 1 ,4-beta- D-glucan celiobiohydrolases or cellulose 1 ,4-beta-celiobiosidases.
  • CBHs hydrolyze the 1 ,4- beta-D-giucosidic linkages from the reducing or non-reducing ends of a polymer containing said linkages.
  • “Cellobiohydrolase i” or “CBH ⁇ act from the reducing end of the cellulose fiber.
  • Cellobiohydrolase H or "CBH ⁇ act from the non-reducing end of the cellulose fiber.
  • Celiobiohydrolases typically have a structure consisting of a catalytic domain and one or more "cellulose-binding domains" or “CBD". Such domains can be located either at the N- or C-terminus of the catalytic domain. CBDs have carbohydrate-binding activity and they mediate the binding of the cellulase to crystalline cellulose and presence or absence of binding domains are known to have a major impact on the processivity of an enzyme especially on polymeric substrates.
  • this polypeptide is further characterized by comprising an amino acid sequence having at least 80 % sequence identity to SEQ ID NO:5, more preferably at least 85 % sequence identity to SEQ ID NO:5, such as at least 90 % % sequence identity to SEQ ID NO:5, and most preferably at least 95 % sequence identity to SEQ ID NO.5.
  • polypeptide having cellobiohydrolase activity as defined above is, in an even more preferred embodiment, further characterized as follows: It is. the polypeptide as defined above, wherein one or more of the amino acid residues of the sequence defined by SEQ ID NO: 5 are modified by substitution or deletion at one or more positions which are preferably selected from
  • the present invention also discloses a polypeptide having cellobiohydrolase activity, which comprises an amino acid sequence with at least 85 % sequence identity to SEQ !D NO: 2. It is preferred that this polypeptide with at leat 85 % sequence identity to SEQ ID NO: 2 is a polypeptide which has also a degree of identity with SEQ ID NO: 5 as given above, such as having at least 60 % (or more, see above) sequence identity with the polypeptide given in SEQ ID NO: 5, and/or any one or more of the more particular identity embodiments of percentage identity to SEQ ID NO: 5 as given in detail above.
  • polypeptide having at least 85 % sequence identity to SEQ ID NO: 2 is an embodiment which is comprised in the invention relating to a polypeptide having at ieast 60 % sequence identity with the polypeptide given in SEQ ID NO: 5.
  • the skilled person will readily recognize the common inventive concept underlying the thermostable variants of SEQ ID NO: 2 and SEQ ID NO: 5.
  • the respective polypeptide comprises an amino acid sequence having at ieast 85 % sequence identity to SEQ ID NO: 2 over a sequence length of 500 amino acid residues.
  • the present invention comprises an amino acid sequence having at Ieast 90 %, or even more preferably of at Ieast Ieast 95% or 98% sequence identity to SEQ ID NO: 2 over a sequence length of 500 amino acid residues.
  • the parental sequence is given in SEQ ID NO: 2.
  • the sequence derives from the C-terminal fusion of the linker domain and cellulose binding domain of Trichoderma reesei CBHI (SEQ ID NO: 4) to the catalytic domain of Talaromyces emersonii CBHI (SEQ ID NO: 5).
  • the invention further comprises other fusion proteins comprising any cellulose binding domain and a derivative of the catalytic domain of Talaromyces emersonii CBHi (SEQ ID NO: 5), preferably with the temperature stability characteristics given above.
  • the cellulose binding domain may be from any source.
  • the polypeptides according to the invention may additionally carry a hexahtstidine tag.
  • variants of any one of the polypeptides shown in SEQ ID NO: 42, 44, 46, 48 or 50 are included in this invention.
  • the variants are preferably such that the polypeptides exhibit temperature stability, as described and defined above.
  • the polypeptide of the present invention preferably comprises an amino acid sequence having at least 90 %, preferably at least 95 %, more preferably at least 99 % sequence identity to SEQ ID NO: 2. Furthermore, it is particularly preferred that the amino acid sequence of the polypeptide has the sequence as defined by SEQ !D NO: 2, or a sequence as defined by SEQ ID NO: 2 wherein 1 to 75, more preferably 1 to 35 amino acid residues are substituted, deleted, or inserted.
  • Protein variants are polypeptides whose amino acid sequence differs in one or more positions from this parental protein, whereby differences might be replacements of one amino acid by another, deletions of single or several. amino acids, or insertion of additional amino acids or stretches of amino acids into the parental sequence.
  • Per definition variants of the parental polypeptide shall be distinguished from other polypeptides by comparison of sequence identity (alignments) using the CiustalW Algorithm (Larkin M.A., Blackshields G., Brown N.P., Chenna R., cGettigan P.
  • amino acids amino acids, peptides, nucleotides and nucleic acids are named according to the suggestions of SUPAC. Generally amino acids are named within this document according to the one letter code.
  • any amino acid residue may be substituted for the amino acid residue present in the position.
  • the alanine may be deleted or substituted for any other amino acid residue (i.e. any one of R, N, D, C, Q, E, G, H, i, L, K, M, F, P, S, T, W, Y and V).
  • amino acid mutation refers to an amino acid mutation that a person skilled in the art would consider similar to a first mutation. Similar in this context means an amino acid that has similar chemical characteristics, if, for example, a mutation at a specific position ieads to a substitution of a non-aiiphatic amino acid residue (e.g. Ser) with an aliphatic amino acid residue (e.g. Leu), then ' a substitution at the same position with a different aliphatic amino acid (e.g. lie or Val) is referred to as a similar mutation.
  • Further amino acid characteristics include size of the residue, hydrophobicity, poiarity, charge, pK- value, and other amino acid characteristics known in the art.
  • a similar mutation may include substitution such as basic for basic, acidic for acidic, polar for poiar etc.
  • the sets of amino acids thus derived are likely to be conserved for structural reasons. These sets can be described in the form of a Venn diagram (Livingstone CD. and Barton GJ. (1993) "Protein sequence alignments: a strategy for the hierarchical analysis of residue conservation” Comput.Appl Biosci. 9: 745-756; Taylor W. R. (1986) "The classification of amino acid conservation” J.Theor.Bio!. 1 19; 205-218).
  • the first glutamine (Q) of the amino acid sequence QQAGTA within the parental protein sequence given in SEQ ID NO: 2 is referred to as position number 1 or Q1 or glutamine 1.
  • the numbering of all amino acids will be according to their position in the parental sequence given in SEQ ID NO: 2 relative to this position number 1.
  • the present invention furthermore discloses specific variants of the polypeptides of the present invention as given above, such as variants of SEQ !D NO: 2, with changes of their sequence at one or more of the positionsgiven hereafter.
  • the invention provides, in a particular embodiment, the polypeptide as above, wherein one or more of the foilowing amino acid residues of the sequence defined by SEQ ID NO: 2 are preferably modified by substitution or deletion at positions Q1 , Q2, G4, A6 ; T7, A8, N10, P12, T15, A21 , G23, S24, T26, T27, Q28, N29, G30, A31 , V32, N37, W40, V41 , G46, Y47, T48, N49, C5G, T52, N54, D57, T59, Y60, D64, E65, A68, Q69, A72, V84, S86, S89, S90, K92, S99, Q109, DU O, D1 1 1 1
  • one or more of the following amino acid residues of the sequence defined by SEQ !D NO: 2 are preferably modified by substitution or deletion at positions selected from Q1 , Q2, G4, A6, T7, A8, N10, A21 , S24, T26, T27, Q28, N29, G30, W40, Y47, D64, E65, A68, Q69, A72, S86, K92, K1 18, Y155, D181 , E183, Q190, S192, N194, D202, H203, P224, T229, G231 , M234, S236, T243, D247, S31 1 , N318, D320, T335, A340, T344, D346, Q349, K355, Y374, A375, T387, D390, T392, T393, Y422, P436, P442, N445, R446, T448, T451 , R453, P462, G463, H468, P480, V
  • Also comprised in the invention are the respective mutations at any one or more of the specified mutations 1 to 430 of SEQ ID NO: 5.
  • residues 1 to 430 of SEQ ID NO: 5 are equivalent to positions 1 to 430 of SEQ ID N02, and can therefore readily transfer the detailed teaching given above and below for preferred modifications of SEQ ID NO: 2. for any one or more of positions 1 to 430 to the respective one or more position (1 to 430) of SEQ ID NQ5.
  • D390 is one .particular position at which a modification in SEQ ID NO: 2 is preferred
  • D390 is likewise a position at which a modification in SEQ ID NO: 5 is preferred.
  • the variant of the polypeptide of the present invention is a polypeptide as described above, wherein specifically one or more of the following amino acid residues of the sequence defined by SEQ ID NO: 2 are modified as shown in Table 1 . Shown are preferred, more preferred and most preferred modification. Any of these mutations may be combined, with each other. However, in a particular embodiment it is preferred that the mutations are selected only among the more preferred and most preferred embodiments shown in Table 1 . Even more preferably, only modifications indicated as most preferred are chosen. The skilled person will be aware that any several such mutations are combineable with each other.
  • the variant of the polypeptides of the present invention as generally defined above comprises in a particular embodiment an amino acid sequence selected from the sequences with mutations with respect to SEQ ID NO: 2, optionally fused with a C- terminal 6x-His Tag, listed in the following Table 2.
  • G4C Q28K, A72C, S86T, E183 , D202N, P224L, S311G, N318Y, D320N, D346A, Q349R, T392M,
  • G4C E65K, A72C, S86T, E183M, D2021, P224L, S311 N, N318Y, D320N, T335I, D346V, Q349R,
  • N445D R446G, H468Q, V482I Q2S, G4C, A6L, T7Q, A8S, N10T, Q28R, E65V, A72C, E183M, P224L, S31 1 G, N318Y, T335I, D346A, Q349K, T393V, Y422F, P442S, N445D,
  • the present invention discloses a nucleic acid encoding the polypeptide of the present invention.
  • the nucleic acid is a polynucleotide sequence (DNA or RNA) which is, when set under control of an appropriate promoter and transferred into a suitable biological host or chemical environment, processed to the encoded polypeptide, whereby the process also includes all post-translational and post-transcriptional steps necessary.
  • the coding sequence can be easily adapted by variation of degenerated base-triplets, alteration of signal sequences, or by introduction of introns, without affecting the molecular properties of the encoded protein.
  • the nucleic acid of the present invention has preferably at least 95 %, more preferably at least 97 %, and most preferably 100% identity to SEQ ID NO: 1.
  • the present invention also provides a vector comprising this nucleic acid and a host transformed with said vector.
  • the present invention also discloses methods for the production of polypeptides of the present invention and variants thereof in various host cells, including yeast and fungal hosts. It also discloses the use of the resulting strains for the improvement of protein properties by variation of the sequence. Furthermore, the present invention discloses methods for the application of such polypeptides in the hydrolysis of cellulose.
  • a further aspect of the invention discloses vectors and methods for the production of protein variants of SEQ ID NO: 2, expressing them in yeast and testing their activity on cellulosic material by measuring the released mono- and/or oiigomeric sugar molecules.
  • the present invention further relates to a method of producing a cellobiohydrolase protein, comprising the steps: a. obtaining a host cell, which has been transformed with a vector comprising the nucleic acid of the present invention;
  • this method of producing a cellobiohydrolase protein is restricted to a method for the production of a celiobiohydrolase protein as provided by this invention, such as having the 1T50 value given above, and/or being one of the specific variants of SEQ ID N02 or SEQ ID NO: 5 as provided with this application and described in detail elsewhere in this specification.
  • the host cell is derived from the group consisting of Saccharomyces, Schizosaccharomyces, Kluyverornyces, Pichia, Hansenula, Aspergillus, Trichoderma, Penicillium, Candida and Yarrowina.
  • the host cell is preferably capable of producing ethanol, wherein most preferred yeasts include Saccharomyces cerevisiae, Pichia stipitis, Pachysoien tannophilus, or a methylotrophic yeast, preferably derived from the group of host cells comprising Pichia methano!ica, Pichia pasto s, Pichia angusta, Hansenula poiymorpha.
  • yeast shall herein refer to all lower eukaryotic organisms showing a unicellular vegetative state in their life cycle. This especially includes organisms of the class Saccharomycetes, in particular of the genus Saccharomyces, Pachysoien, Pichia, Candida, Yarrowina, Debaromyces, Klyveromyces, Zygosaccharomyces.
  • one aspect of the invention relates to the expression of the claimed polypeptide and variants thereof in yeast.
  • the efficient expression of this fusion protein (SEQ ID NO: 2) and derivative protein variants of SEQ ID NO: 2 from yeast can be achieved by insertion of the nucleic acid molecule of SEQ ID NO: 1 starting from nucleotide position 1 into an expression vector under control of at least one appropriate promoter sequence and fusion of the nucleotide molecule to an appropriate signal peptide, for example to the signal peptide of the mating factor alpha of Saccharomyces cerevisiae.
  • the polypeptide of the present invention and variants thereof are expressed and secreted at a level of more than 100 mg/l, more preferably of more than 200 mg/l, particularly preferably of more than 500 mg/l, or most preferably of more than 1 g/l into the supernatant after introduction of a nucleic acid encoding a polypeptide having an amino acid sequence with at least 85% sequence identity to the SEQ ID NO: 2 into a yeast.
  • the cultivation and isolation of the supernatant can be carried out as described in Example 3.
  • a further aspect the invention discloses methods for the production of a polypeptide according to the present invention in a filamentous fungus, preferably in a fungus of the genus Aspergillus or Trichoderma, more preferably in a fungus of the genus Trichoderma, most preferably in Trichoderma reesei "Filamentous fungi" or “fungi” shall herein refer to all lower eukaryotsc organisms showing hypha! growth during at least one state in their life cycle.
  • the polypeptide is expressed by fusion of the coding region of a compatible signal sequence to the nucleic acid molecule starting with nucleotide position 52 of SEQ ID NO: 3, as it was done in SEQ ID NO: 3 with the signal sequence of the Trichoderma reesei CBH1, and the positioning of the fusion peptide under control of a sufficiently strong promoter followed by transfer of the genetic construct to the host ceil. Examples for such promoters and signal sequences as well as techniques for an efficient transfer have been described in the art.
  • the present invention further relates to a method for identifying a polypeptide or polypeptides having cellobiohydrolase activity, comprising the steps of: a. Generating a library of mutant genes encoding mutant proteins by mutagenesis of a nucleic acid according to claim 9 or a nucleic acid having the sequence defined by SEQ ID NO: 6 (encoding SEQ !D NO: 5), preferably having the sequence defined by SEQ ID NO: 1 ;
  • step d. may be performed by utilizing a well-plate format.
  • This format preferably allows the high-throughput performance of the method for identifying polypeptides having cellobiohydrolase activity.
  • this method for identifying polypeptides having cellobiohydrolase activity is restricted to a method, wherein the polypeptide(s) having cellobiohydrolase activity is one or more polypeptide(s) as provided by this invention, such as having the ⁇ 50 value given above, and/or being one of the specific variants of SEQ ID N02 or SEQ ID NO: 5 as below.
  • the steps e. to g. of the method for identifying polypeptides having celiobiohydrolase activity are performed as follows: e. Incubating the expressed mutant protein with celiuiosic material;
  • the method for identifying polypeptides having celiobiohydrolase activity comprises the additional steps of: h. Sequencing the selected mutant gene or protein;
  • the method is further characterized by measuring the IT50 value of the obtained polypeptide.
  • the IT50 value may be measured as described in the examples below.
  • this may be followed by a step of selction of those polypeptides, which display the a desired IT 50 value, such as at least 60 °C, at least 62 °C and the like,
  • the method is suitable for identifying polypeptides exhibiting celiobiohydrolase activity and an elevated IT50 value, i.e. thermostable polypeptides with celiobiohydrolase activity.
  • the present invention further provides a method of preparing a polypeptide having celiobiohydrolase activity, comprising the steps: a. Providing a polypeptide having celiobiohydrolase activity comprising an amino sequence having at ieast 54 % sequence identity to the catalytic domain of SEQ ID NO: 2 (SEQ ID NO: 5) (such as preferably, at ieast 60 %, at Ieast 62 %, at Ieast 64 %, at Ieast 66, %, at Ieast 68 % or at Ieast 70 %, whereby at Ieast 68 % or at Ieast 70 % are the most preferred embodiments);
  • step c Preparing a mutant polypeptide of the polypeptide provided in step a. by carrying out the amino acid modification(s) identified in step b. through site- directed mutagenesis.
  • the polypeptide provided in step a. of the method of preparing a polypeptide having cellobiohydrolase activity is a wild type cellobiohydrolase derived from Trichoderrna reesei.
  • the present invention further provides polypeptides having cellobiohydrolase activity, which are obtainable by the method of preparing a polypeptide having cellobiohydrolase activity according to the present invention.
  • the present invention provides a composition comprising a polypeptide and/or variants thereof of the present invention and one or more cellulases, e.g. one or more endoglucanases and/or one or more beta-glucosidases and/or one or more further cellobiohydrolases and/or one or more xylanases.
  • Cellulases or “Cellulolytic enzymes” are defined as enzymes capable of hydroiysing celluiosic substrates or derivatives or mixed feedstocks comprising celluiosic polymers. Such enzymes are referred to as having "cellulolytic activity", thus being able to hydrotyze cellulose molecules from such material into smaller oligo- or monosaccharides.
  • Cellulolytic enzymes include cellulases and hemicellulases, in particular they include cellobiohydrolases (CBHs), endoglucanases (EGs) and beta-glucosidases (BGLs).
  • the present invention further provides a polypeptide having cellobiohydrolase activity, wherein the poiypepttde comprises an amino acid sequence having at least 80 %, preferably at least 95%, more preferably at least 98%, even more, preferably at least 99%, and most preferably 99, 6 % sequence identity to SEQ ID NO: 5,.
  • such a polypeptide is a polypeptide wherein one or more of the following amino acid residues of the sequence defined by SEQ ID NO: 5 are modified by substitution or deletion of: Q1 , Q2, G4, A6, T7, A8, N10, P12, T15, A21 , G23, S24, T26, T27, Q28, N29, G30, A31 , V32, N37, W40, V41 , G46, Y47, T48, N49, C50, T52, N54, D57, T59, Y60, D64, E65, A68, Q69, A72, V84, S86, S89, S90, K92, S99, Q109, D1 10, DU1 , 11 16, F1 17, K1 18, L1 19, L120, D129, V130, G139, A145, 146, V152, K154, Y155, N157, N158, K159, K163, G167, Q172, F179, 1180,
  • the polypeptide having ceilobiohydroiase activity with an amino acid sequence having at least 80 % sequence identity to SEQ ID NO: 5 comprises one or more modified amino acid residues of the sequence defined by SEQ !D NO: 5:
  • the polypeptide given in SEQ ID NO: 5 may, by means of example, be modified as follows: Q1 L, G4, A6G/V, T15S, Q28Q/R, W40R, D64N, E65K/V, A72V, .
  • the polypeptide .having ceilobiohydroiase activity comprises one or more modified amino acid residues of the sequence defined by SEQ !D NO: 5 as indicated in the following Table 3.
  • SEQ iD NO: 2 two or more of such specific modifications may be combined with each other, such as preferably two or more of the more preferred or most preferred modifications may be combined with each other, and, which is particularly preferred, two pr more of the most preferred modifications according to Table 3 may be combined with each other.
  • polypeptide as defined above, further characterized by comprising a modification of SEQ !D NO: 5, which is a specific modification as given in the following Table 3a.
  • SEQ !D NO: 5 which is a specific modification as given in the following Table 3a.
  • Each of these polypeptides defines a mutant version of the polypeptide given in SEQ ID NO: 5.
  • G4C, Q28R, E65M [ A72C, S86T, E183K. S192I, H203R, S311N, D346E,

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